Microbial contamination by, for example, Gram positive bacteria, Gram negative bacteria, yeast, fungi, and molds may cause severe illness and, in some cases, even death in humans. Manufacturers in certain industries, for example, the pharmaceutical, medical device, water, and food industries, must meet exacting standards to verify that their products do not contain levels of microbial contaminants that would otherwise compromise the health of the recipient. These industries require frequent, accurate, and sensitive testing for the presence of such microbial contaminants to meet certain standards, for example, standards imposed by the United States Food and Drug Administration (USFDA) or the Environmental Protection Agency. By way of example, the USFDA requires certain manufacturers of pharmaceuticals and invasive medical devices to establish that their products are free of detectable levels of Gram negative bacterial endotoxin.
To date, a variety of assays have been developed to detect the presence and/or amount of a microbial contaminants in a test sample. One family of assays use hemocyte lysates prepared from the hemolymph of crustaceans, for example, horseshoe crabs. These assays typically exploit, in one way or another, a clotting cascade that occurs when the hemocyte lysate is exposed to a microbial contaminant. For example, FIG. 1 shows a schematic representation of certain clotting cascades known to be present in hemocyte lysate produced from the hemolymph of the horseshoe crab, Limulus polyphemus. Such lysates are known in the art as Limulus amebocyte lysate or LAL.
As shown in FIG. 1, the coagulation system of LAL, like the mammalian blood coagulation system, comprises at least two coagulation cascades that include an endotoxin or lipopolysaccharide (LPS) mediated pathway (the Factor C pathway) and a (1→3)-β-D glucan mediated pathway (the Factor G pathway). See, for example, Morita et al. (1981) FEBS LETT. 129: 318-321; and Iwanaga et al. (1986) J. PROTEIN CHEM. 5: 255-268.
It is understood that Gram negative bacteria can be detected using LAL based assays. For example, Gram negative bacteria produce endotoxin or LPS, which after binding to LPS binding protein activates the Factor C pathway in LAL (see, FIG. 1). The endotoxin or LPS-mediated activation of LAL is well understood and has been thoroughly documented in the art. See, for example, Levin et al. (1968) THROMB. DIATH. HAEMORRH. 19: 186; Nakamura et al. (1986) EUR. J. BIOCHEM. 154: 511; Muta et al. (1987) J. BIOCHEM. 101: 1321; and Ho et al. (1993) BIOCHEM. & MOL. BIOL. INT. 29: 687. When bacterial endotoxin is contacted with LAL, the endotoxin initiates a series of enzymatic reactions, known as the Factor C pathway, that are understood to involve three serine protease zymogens called Factor C, Factor B, and pro-clotting enzyme (see, FIG. 1). Briefly, upon exposure to endotoxin, the endotoxin-sensitive factor, Factor C, is activated. Activated Factor C thereafter hydrolyses and activates Factor B, whereupon activated Factor B activates proclotting enzyme to produce clotting enzyme. The clotting enzyme thereafter hydrolyzes specific sites, for example, Arg18-Thr19 and Arg46-Gly47 of coagulogen, an invertebrate, fibrinogen-like clottable protein, to produce a coagulin gel. See, for example, U.S. Pat. No. 5,605,806.
Furthermore, it is also understood that (1→3)-β-D glucans and other LAL-reactive glucans, produced by microorganisms such as yeasts and molds, can also activate the clotting cascade of LAL, through a different enzymatic pathway known as the Factor G pathway (see, FIG. 1). It is understood that Factor G is a serine protease zymogen that becomes activated by (1→3)-β-D glucan or other LAL reactive glucans. Upon exposure to (1→3)-β-D glucan, for example, Factor G is activated to produce activated Factor G. It is understood that activated Factor G thereafter converts the proclotting enzyme into clotting enzyme, whereupon the clotting enzyme converts coagulogen into coagulin.
Presently, LAL is employed as the amebocyte lysate of choice in many assays for detecting the presence of Gram negative bacteria, fungus or molds because of its sensitivity, specificity, and relative ease for avoiding interference by other components that may be present in a sample. For example, LAL, when combined with a sample containing bacterial endotoxin and optionally with certain LAL substrates, reacts with the endotoxin in the sample to produce a detectable product, such as a gel, increase in turbidity, or a colored or light-emitting product in the case of a synthetic chromogenic substrate. The resulting product may be detected, for example, either visually or by the use of an optical detector.
In contrast, assays of comparable sensitivity and specificity for detecting and/or quantifying Gram positive bacterial contaminants have been more difficult to develop. One assay for detecting Gram positive bacterial contamination, known as the In Vitro Pyrogen Test (IPT), is available from Charles River Laboratories (Wilmington, Mass.). The IPT assay is an in vitro alternative to the rabbit pyrogen test, and is an ELISA-based assay that uses fresh or cryoproserved human whole blood. When exposed to pyrogens, immune cells within the whole blood produce interleukin-1β that is detected in the ELISA assay. The IPT assay, however, does not selectively detect the presence of Gram positive bacteria, as it is activated by pyrogens present in Gram positive bacteria, Gram negative bacteria, yeasts and viruses.
Although the detection of bacterial, yeast and fungal contamination can be extremely important, the ability to discriminate between these different organisms can provide useful information about an infectious agent causing an infection in an individual or the source and type of contamination present in a test sample. For example, once an infectious agent has been identified, a physician can then prescribe the most appropriate medication for treating an infection. Furthermore, once the type of bacterial, yeast or fungal contamination has been identified, then this type of information may speed up the process of identifying the source of contamination in, for example, a water supply. As a result, once the source of contamination has been identified, further contamination can be mitigated. Although methods and compositions currently are available for specifically detecting Gram negative bacteria, yeasts, and molds, there is still an ongoing need for further methods and compositions for specifically detecting Gram positive bacteria in a sample of interest.